Conserved DNA Motifs,Including the CENP-B Box-like,Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.     

Halobacterial megaplasmids are negatively supercoiled

Several covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal-field alternating gel electro-phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.     

Detection of Infectious Astroviruses in Water

A method based on the infection of CaCo-2 cells and molecular hybridization with a specific cDNA probe has been developed for the detection of infectious astroviruses in environmental samples. By this procedure wild-type astroviruses have been detected in water from an area where a concurrent gastroenteritis outbreak was reported.     

Colloidal Gold Cytochemistry of Endo-1,4-beta-Glucanase, 1,4-beta-D-Glucan Cellobiohydrolase, and Endo-1,4-beta-Xylanase: Ultrastructure of Sound and Decayed Birch Wood

Colloidal gold coupled to endo-1,4-beta-glucanase II (EG II) and 1,4-beta-D-glucan cellobiohydrolase I (CBH I), isolated from Trichoderma reesei (QM9414), and endo-1,4-beta-xylanase from Aureobasium pullulans (NRRLY-2311-1) was used successfully to determine the ultrastructural localization of cellulose and xylan in sound birch wood. In addition, these enzyme-gold complexes demonstrated the distribution of cellulose and xylan after decay by three white rot fungi, Phanerochaete chrysosporium, Phellinus pini, and Trametes versicolor, and one brown rot fungus, Fomitopis pinicola. Transverse sections of sound wood showed that EG II was localized primarily on the S(1) layer of the secondary wall, whereas CBH I labeled all layers of the secondary wall. Oblique sections showed a high concentration of gold labeling, using EG II or CBH I. Preference for the sides of the microfibrillar structure was observed for both EG II and CBH I, whereas only CBH I had a specificity for the cut ends of microfibrils. Labeling with the xylanase-gold complex occurred primarily in the inner regions of the S(2) layer, S(1), and the middle lamella. In contrast, little labeling occurred in the middle lamella with EG II or CBH I. Intercellular regions within the cell corners of the middle lamella were less electron dense and labeled positively when EG II- and xylanase-gold were used. Wood decayed by P. chrysosporium or P. pini was delignified, and extensive degradation of the middle lamella was evident. The remaining secondary walls labeled with EG II and CBH I, but little labeling was found with the xylanase-gold complex. Wood decayed by T. versicolor was nonselective, and erosion of all cell wall layers was apparent. Remaining wall layers near sites of erosion labeled with both EG II and CBH I. Erosion troughs that reached the S(1) layer or the middle lamella had less xylanase-gold labeling in the adjacent cell wall that remained. Brown-rotted wood had very low levels of gold particles present in sections treated with EG II or xylanase. Labeling with CBH I had the lowest concentrations in the S(2) layer near cell lumina and corresponded to sites with the most extensive degradation.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain

We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N-terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N-terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction.     

Presence of double‐stranded RNAs and virus‐like particles in the entomopathogenic fungus Metarhizium anisopliae

Nucleic acids from 41 strains of Metarhizium anisopliae, obtained from different parts of the world were extracted and examined by electrophoresis. Strong bands of double‐stranded RNA (dsRNA) were detected in two isolates from Brazil, V215 and V291, which had, respectively, 13 and 9 distinct bands ranging in size from ca. 0.75 to 3.5 kb. Icosahedral virus‐like particles (VLPs) (ca. 33 nm in diameter) were observed by transmission electron microscopy in extracts of these isolates. The VLPs and dsRNA were both absent from a clone of the isolate V291 which had been subcultured successively on solid medium. Bioassays against the aphid Myzus persicae showed no detectable difference in virulence between the clone of V291 which contained dsRNA and the clone that did not.     

Erk1/2 signaling mediates the HGF-induced protection against ethanol and acetaldehyde-induced toxicity in the pancreatic RINm5F cell line

Alcohol-induced pancreas damage remains as one of the main risk factors for pancreatitis development. This disorder is poorly understood, particularly the effect of acetaldehyde, the primary alcohol metabolite, in the endocrine pancreas. Hepatocyte growth factor (HGF) is a protective protein in many tissues, displaying antioxidant, antiapoptotic, and proliferative responses. In the present work, we were focused on characterizing the response induced by HGF and its protective mechanism in the RINm5F pancreatic cell line treated with ethanol and acetaldehyde. RINm5F cells were treated with ethanol or acetaldehyde for 12 h in the presence or not of HGF (50 ng/ml). Cells under HGF treatment decreased the content of reactive oxygen species and lipid peroxidation induced by both toxics, improving cell viability. This effect was correlated to an improvement in insulin expression impaired by ethanol and acetaldehyde. Using a specific inhibitor of Erk1/2 abrogated the effects elicited by the growth factor. In conclusion, the work provides mechanistic evidence of the HGF-induced-protective response to the alcohol-induced damage in the main cellular component of the endocrine pancreas.